SMAD4 mutations identified in Iranian patients with colorectal cancer and polyp

Aim: Search for SMAD4 mutations in Colorectal cancer (CRC) or polyp in Iran. Background: Colorectal cancer is one of the five prevalent cancers among the Iranian population; however, its molecular mechanisms are not fully understood. The vast majority of CRCs arise from neoplastic polyp Methods: Colorectal cancer and polyp lesions with matched normal tissues from patients who had undergone colonoscopy in Taleghani Hospital (January 2009 – November 2010) were included in the study. DNA extraction and PCR-sequencing for exons 5-11 of the SMAD-4 gene were carried out on 39 and 30 specimens of polyp and adenocarcinoma, respectively. Results Of cancer and polyp specimens, 33.3% and 28.2%, respectively, were mutated in the Smad-4 gene. The majority of SMAD4 mutations, especially in the MH2 domain were missense mutations (63.6% and 68.75, respectively). In cancer, codon 435 and in polyp, codons 435 and 399 were the most common alterations. Unlike cancer specimens, transversion was found frequently in the polyp (56.25% vs. 35.7%). CG>TA transition was about 18.75% and 14.3% in cancer and polyp samples, respectively. Mutations of codon 264 and C.483-4 were seen both in cancer and neoplastic polyps. Conclusion: As frequent alterations, missense mutations are presumably selected during tumorigenesis and polyposis due to their structural impacts on SMAD4 functions and TGF-ß signaling pathway. The lower frequency of CG>TA can be attributed to global genome hypomethylation. Presumably, SMAD4 mutations had occurred in the primary polyps, and some of these mutated cells then developed into carcinoma. On the other hand, polyp-specific mutations may lower the risk of CRC.


Introduction
The first specified substrate of TGF-β receptor kinases is the proteins of the SMAD family (1). Through their phosphorylation and activation by transmembrane receptor with serine-threonine kinase interaction with regulatory proteins. Moreover, through intramolecular interaction, the MH1 domain suppresses the biological and transcriptional activities of the MH2 domain (8). The majority of SMAD4 alterations cluster in the MH2 domain and often alter residues in the vicinity of protein interface mediating SMAD4 heterooligomerization (9). Mutations at the MH1 domain have been reported to enhance interactions with the MH2 domain (10) and alter DNA binding (11), protein stability, and prevent nuclear translocation (12). SMAD4 is the subject of inactivating mutations in some cancers, and loss of SMAD4 expression is a notable feature of most human cancers (3), including colorectal cancer (CRC) (13). SMAD4 mutations were reported in 2.1-31% of colorectal cancer cases (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Nevertheless, previous studies have established some associations between SMAD4 mutation and protein expression with the survival of patients and progress of colorectal cancer (24). CRC is the second and third most common and lethal cancer in males and females, respectively, worldwide, and more than 1.8 million new cases and 881,000 deaths were estimated to have occurred in 2018 (25). Among Iranian males and females, CRC is one of the five most common cancers (26, 27), accounting for approximately 6.3% of all cancer deaths; 3641 new cases and 2262 deaths from CRC are estimated annually (28). While the incidence rate of colorectal decreased annually in the USA during 1975-2017 (29), the rate is rapidly increasing in several regions historically at low risk (30) and in younger generations of Iran (31). There are several pathways for CRC (32). The vast majority of CRCs arise from precursor lesions, termed polyps (33), and the adenoma-carcinoma sequence accounts for nearly 95% of all CRCs (34). Moreover, 15-20% of sporadic CRC develops from serrated polyps through pathways distinct from the traditional adenoma-carcinoma sequence (35). Given the earlier studies on the importance of SMAD4 integrity and considering the prevalence of CRC in Iran, this study was designed to evaluate the contribution of SMAD4 mutations in colorectal carcinogenesis and polyposis and their correlation with clinicopathological aspects. To date, no attempt has been made to search for SMAD4 mutations in CRC or polyp in Iran.

Patients
Colorectal cancer (intestinal-type) and polyp lesions with matched normal tissues were collected from patients who had undergone a colonoscopy of the gastrointestinal tract in Taleghani Hospital (January 2009 -November 2010, Tehran, Iran). After resection, the specimens were immediately processed for the DNA extraction or were frozen at -80 °C until extraction. Specimens were obtained under informed consent and the patients were considered competent to decide to enroll. This study was approved by the Ethics and Scientific Committee of our institution following the principles of the Declaration of Helsinki. The samples were histologically diagnosed by pathologists as being CRC and polyp; only samples containing at least 80% tumor nuclei were selected for DNA extraction.

Sequence analysis for mutations detection
In search of nucleotide alterations of the SMAD4 gene in exons 5, 6, and 8-11, PCR sequencing was carried out using primers as presented in Table 1. Primers were designed based on GenBank sequence NG_013013.2 (GI: 383387807). PCR reactions containing 10 pmol of each primer, 200 mM of each dNTP, and 0.5 U Taq polymerase were conducted in the cycling program, as shown in Table 1. Considering that for all reactions, the initial denaturation and final extension were 5 minutes at 94 and 72 for 10 minutes, respectively. DNA sequencing was performed using the ABI3130X Genetic Analyzer. To distinguish somatic mutations from germline mutations, the mutant sequences from tumor/polyp were compared with the sequence of DNA extracted from blood leucocytes of the same person. Statistical analysis SPSS 20.0 was used for statistical analyses. The association of SMAD4 nucleotide alterations and clinical parameters, such as location and histological type of polyps, was evaluated using the Fisher exact test. A p-value< 0.05 was regarded as statistically significant.

Patient characteristics
In this study, 39 and 30 fresh tissue specimens for colorectal polyp and intestinal-type adenocarcinoma, respectively, and adjacent normal tissue were examined for the desired sequences of the SMAD4 gene. The characteristics of patients are given in Table 2.
In cancer tissues, 14 mutations were detected in the coding regions and intronic region of the SMAD4 gene. In the coding regions, most of the mutations clustered in the MH2 domain (7 missense mutations out of 11 = 63.6%), and the remainder (36.4%) were mapped to the linker region including two missense and two silent mutations. Overall, nine missense (64.3%), two silent (14.3%), and three intronic (21.4%) mutations were identified in cancer. Missense mutations at codon 435 (ATA>GTA) were the most frequent mutation (5/14, 35.7%) in cancer patients ( Figure 1A).

Type of mutations
In polyp samples, transversion was the most frequent substitution (56.25% vs. 43.75%) while in the  (Table 4).

Mutations and types of polyp compared with cancerous mutations
The frequency of mutations in each type of polyps is shown in Table 5. Hyperplastic, tubular, and serrated polyps were the most mutated samples: 36.4%, 27.3%, and 18.2% of total mutations, respectively. Moreover, all serrated polyps (2/2) and half of the hyperplastic polyps were mutated. One serrated polyp had four mutations at codons 361, 386, 399, and 435 (Table 5).

Mutations and locations of specimens
As shown in Table 7, most cancer and polyp samples were obtained from the rectum and colon, respectively. In cancer samples, 80% of detected mutations occurred in the rectum, while in polyps, 81.9% of mutations were identified in the colon. In other words, in cancer samples, 40% and 20% of rectum and colon specimens, respectively, were mutated, while in polyps, the percentage of mutated specimens was not different (28.1% vs. 28.6%). The association between location and mutation is considered to be statistically significant (Fisher's exact test: two-tailed p-value = 0.0089).

Mutations, age, and gender
Age at diagnosis and gender were not statistically different between patients with and without mutation in both polyp and cancer samples.

Discussion
Colorectal cancer accounted for about 10% of cancer cases and deaths worldwide in 2018 (25). However, the molecular mechanisms of CRC remain to be elucidated. To reveal some aspects of this matter, the current study was designed to evaluate the contribution of SMAD4 alterations in colorectal carcinogenesis. In the present study, somatic SMAD4 mutations were found in 33.3% and 28.2% of analyzed specimens with CRC and polyp, respectively. To date, varying rates of SMAD4 mutations in CRC have been reported. Based on previous reports, 2.1%-31% of CRC samples may be mutated at the SMAD4 gene (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). It has been shown that mice with SMAD4 deletion or loss of SMAD4-dependent signaling have increased susceptibility to developing colorectal polyp and cancer (36). On the other hand, alterations of SMAD4 have been associated with both metastasis (19) and a significantly poor prognosis (20). The loss of SMAD4 function causes an increased genomic instability in epithelial tumors, blocks growth inhibition and apoptosis which are normally induced by TGF-β, and promotes inflammation through TGF-β, thereby possibly paving the way for the expansion of  genetically defected cells during polyposis and tumorigenesis (37). Considering that SMAD4 gene is located at 18q21, a region where allelic loss is very prevalent in CRC (38,39), SMAD4 may play an important role as a tumor suppressor gene, and genetic alterations may have some role in silencing SMAD4 in (a fraction of) CRC (40).
In accordance with previous works, the majority of mutations clustered in the MH2 domain in both polyp and cancer, while the MH2 domain represents only 41.5% of the coding sequence (41). MH2 residues are necessary for homodimerization and heterooligomerization with SMAD 2 or 3 proteins (42). Therefore, mutations in this region may cause a cessation of signal transmission through the TGF-ß pathway, which has been connected to many human diseases such as cancer (43).
In agreement with previous reports on CRC (19,22,41), missense mutations appeared to occur more frequently. Missense mutations are presumably selected for/during tumorigenesis due to their structural impacts on a specific function (44) or locking a protein in a specified state. They can also lead to drastic destabilization of the mutant protein or alter protein binding properties and its interaction network (45,46). For example, the majority of missense mutations outside of codons 330-370 inactivate SMAD4 through protein degradation (47). However, the extent to which cancer mutations might affect biomolecular structure and interactions remains unknown. Using structurebased methods may be helpful to predict the effects of mutations on protein stability and protein-protein interactions (48). The rate of transition in cancer specimens was higher than transversion (64.3% vs. 35.7%). The situation was reversed in polyp samples (43.75% vs. 56.25%). The observed difference suggests that the mechanisms causing SMAD4 mutations in CRC and polyp are somewhat distinct from each other, or maybe conversion and transition of adenoma into early carcinoma needs different engines (19). The rate of CG>TA transition was low in both polyp and cancer compared to previous reports (approximately 54%) in colorectal tumors (19). CG>TA transition is thought to result from hydrolytic deamination of 5-methylcytosine residues particularly at the CpG dinucleotide in the body of genes, outside of CpG islands. Therefore, the lower frequency of CG>TA transitions can be attributed to the global genome hypomethylation as a key initiating event in cancer development (49). The current authors' previous work on gastritis lesions showed that global genome hypomethylation may induce a different pattern (50) and spectrum of mutations of the p53 gene in an Iranian population (51), which implies other mechanism(s) in cancer development in the Iranian population. This study detected some identical and several exclusive mutations in CRC and neoplastic polyps. The presence of the same SMAD4 mutations in both CRC and neoplastic polyp (264, 386, 435, c.484-4) suggests that these mutations had occurred in the primary polyps, and then the cell population having these mutations gained the potential and permission to develop into carcinoma. Therefore, these types of mutations have specific advantages for polyposis or carcinogenesis and can be used as diagnostic or prognostic markers. On the other hand, polyp-specific mutations (242, 361, 399, c.248+64) may lower the risk of transformation of these polyps toward CRC.
To summarize, SMAD4 alterations in CRC and polyp were investigated. The current findings showed some previously reported as well as some novel mutations. These mutations may result in the loss of multiple functional properties of SMAD4, such as communication network (homodimerization, heterooligomerization), subcellular localization, transcriptional activation, and altered stability compared with wild type protein, and such switching may contribute to tumorigenesis. However, their functional consequences must be evaluated. Due to limited access to polyp samples, especially cancerous polyps, the findings of the current study should be validated in a larger population.